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Protein engineering. Tailor-made biocatalysts The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity

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protein engineering
Protein engineering

Tailor-made biocatalysts

  • The efficient application of biocatalysts requires the availability of suitable enzymes with high activity and stability under process conditions, desired substrate selectivity and high enantioselectivity
  • Rational (re)design versus directed evolution
protein engineering1
Protein engineering

Genetic manipulation techniques

  • Large-scale supply of enzymes at reasonable price
  • Identification of new biocatalysts (screening) doesnot always yield suitable enzymes for a given synthetic problem
  • Computer-aided site-directed mutagenesis
  • Directed (molecular) evolution
protein engineering2
Protein engineering

Site-directed mutagenesis

  • Requires structural information and knowledge about relationship between sequence, structure, function and mechanism
  • Very information-intensive
  • Rapid progress in NMR / X-ray methods
  • Genome sequence information
  • Molecular modeling, bioinformatics
  • Prediction of selectivity, activity, stability etc.
protein engineering3
Protein engineering

Rational redesign strategy

  • Protein structure
  • Planning of mutants, SDM
  • Vectors containing mutated genes
  • Transformation in E. coli
  • Protein expression and purification
  • Mutant enzyme analysis
  • Negative mutants
  • Improved mutant enzymes
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Protein engineering

Rational redesign

  • Amino acid substitutions often selected by sequence comparison with homologous sequences
  • Results have to be carefully interpreted
  • Minor changes by a single point mutation may cause significant structural disturbance
  • Comparison of 3D-structure of mutant and wild-type enzyme necessary
protein engineering5
Protein engineering

Inversion of stereospecificity of VAO

  • Current Opinion in Chemical Biology (2001)
  • A very nice study on alteration of enantioselectivity based on structural comparison of two members of structurally related FAD-dependent oxidoreductases, of which one is (R)-specific and the other (S)-specific
  • Site-directed mutagenesis introduced (S)-selectivity in the (R)-selective wild-type enzyme
  • Structural analysis of the mutant enzyme revealed that the mutations are really site-directed
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Protein engineering

Directed evolution

  • Evolutive biotechnology, molecular evolution
  • Random mutagenesis of the gene encoding the biocatalyst (e.g. by error-prone PCR)
  • DNA shuffling: recombination of gene fragments (staggered extension process or random priming recombination)
protein engineering7
Protein engineering

Directed evolution strategy

  • Random mutagenesis
  • Library of mutated genes
  • Transformation in E. coli
  • Mutant library > 10.000 clones
  • Protein expression in microtiter plates
  • Selection parameters
  • Mutant enzyme and product analysis
  • In vitro-recombination, transformation etc.
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Protein engineering

Selection parameters

  • Substrate range
  • Stability in organic solvent
  • Stability towards reaction conditions
  • Thermal stability
  • High-throughput product analysis
  • Robot technology
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Protein engineering

Selection parameters

  • Hydrolysis of esters: agar-plate assay based on pH indicators
  • Parallel assaying of replica-plated colonieswith substrate analog
  • Isotopically labeled substrates
  • Capillary electrophoresis (7000 samples per day)
  • Optimization with saturation mutagenesis
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Protein engineering

Digital image screening

  • Naphthalene hydroxylation by P450cam
  • Co-expression of horseradish peroxidase
  • Fluorescent products amenable by digital screening
  • P450 hydroxylation of indole to indigo
  • Inversion of enantioselectivity
  • Increase of peroxidase specificity with guaiacol
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Protein engineering

Improving thermostability

  • Cold-adapted proteases
  • Combined screening for activity, thermostability, organic solvent tolerance and pH-profile
  • Engineering of entire metabolic pathways
  • Phytoene desaturase and lycopene cyclase shuffling for carotenoid biosynthesis
  • Molecular breeding
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Protein engineering

Biochemistry

Vanillyl-alcohol oxidase

  • Production of natural vanillin

2-Hydroxybiphenyl monooxygenase

  • Large-scale production of substituted catechols

Galactose oxidase

  • Production of new oligosaccharides
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